Interpretive Summary: Vegetables are essential components of the human diet, particularly because they provide phytonutrients including vitamins, minerals and fiber. However, the contents of essential phytonutrients is much below the threshold to meet daily requirements. The focus of our research is on using genetic engineering of key, beneficial genes and testing what changes they bring about when introduced into tomato plant. We have developed transgenic lines with stable integration of a gene critical for the biosynthesis of polyamines called spermidine and spermine. To obtain an account of the changes in their metabolites as compared to the wild type plants, we embarked on using nuclear magnetic resonance (NMR) spectroscopy to profile the metabolites in conjunction with real time quantification of key gene transcripts. Resonances found in the genetically engineered tomato fruit samples were not different than those in the wild type. Fruits were found to sense the increase in the amines as organic-N, which resulted in enhanced synthesis of biomolecules, increased acid to sugar ratio - a good attribute for the fruit flavor, and in the accumulation of another 'vital amine' called choline, which is essential as a micronutrient for brain development. This research is of interest to plant and animal biologists and biotechnologists who are interested in understanding the basic metabolism and key processes involved in the accumulation of phytonutrients in order to develop strategies for improving nutritional quality, vine and shelf life.

Technical Abstract:
Polyamines are ubiquitous aliphatic amines that have been implicated in myriad processes but their precise biochemical roles are not fully understood. We have carried out metabolite profiling analyses of transgenic, tomato fruit engineered to accumulate higher polyamines, spermidine (Spd) and spermine (Spm) (Mehta et al., Nature Biotechnol. 20: 613-618, 2002), using NMR spectroscopy, to bring an insight into the metabolic processes that Spd/Spm regulate in plants. Distinct metabolites were found to accumulate (Glu, Gln, Asn, choline, and three unidentified compounds) or decrease (sucrose, glucose, Asp) in parallel to Spd/Spm accumulation in the transgenic versus the control (wildtype and azygous) fruits. The pathways involved in the nitrogen sensing/signaling and carbon metabolism seem preferentially activated in the high Spd/Spm transgenics. Statistical treatment of the metabolite variables distinguished the control fruits from the transgenic fruit and provided credence to the pronounced, differential metabolite profiles seen during later stages of fruit ripening in the transgenics. The metabolite profiling analysis strikingly reveal that Spd/Spm are perceived as nitrogenous compounds by the fruit cells, which in turn stimulate carbon sequestration. This is seen manifested in higher respiratory activity and upregulation of PEP carboxylase and NADP-dependent isocitrate dehydrogenase transcripts in the transgenic fruit compared to controls indicating high metabolic status of the transgenics even late in fruit ripening. The metabolite profiling analysis has unraveled important consequences of higher threshold of Spd/Spm in a fruit, including previously little known facets such as N:C interdependent signaling involving subcellular compartments.